In the past decade, biomolecule detection has become increasingly important in biological studies and clinical applications. In particular, the expressions of lipids, proteins and nucleotides are widely adopted as criteria in disease diagnosis and management, which require sensitive and quantitative determination of the level of these molecular markers (Zangar, R. C. et al., Expert Rev. Proteomics, 3:37-44 (2006); Schupbach, J., AIDS Rev., 4:83-92 (2002)). Traditional detection assays relying on small-molecule reporters such as radioisotopes and fluorophores have found little use in these applications because of poor stability, low sensitivity and difficult sample processing. The need for simple and reliable detection methods has led to the development of various inorganic nanoparticle-based probes. With remarkable magnetic, optical or plasmonic properties, these nanoparticles have greatly expanded the scope of specific bioassays (Peng, H. I. and Miller, B. L., The Analyst, 136:436-447 (2011); Alivisatos, P., Nat. Biotechnol., 22:47-52 (2004); Dykman, L. and Khlebtsov, N., Chemical Society Reviews, 41:2256-2282 (2012)). For instance, quantum dots and gold nanoparticles can drastically increase the sensitivity of immunofluorescence staining, microarrays and microfluidics (Nam, J. M. et al., Science, 301:1884-1886 (2003); Nam, J. M. et al., J. Am. Chem. Soc., 126:5932-5933 (2004); Chin, C. D. et al., Nat Med, 17:1015-1019 (2011)). Magnetite and maghemite nanoparticles are being utilized for on-site detection of proteins, nucleotides and bacteria (Perez, J. M. et al., Nat. Biotechnol. 20:816-820 (2002)), in addition to enriching target molecules in detection assays (Nam, J. M. et al., Science, 301:1884-1886 (2003); Lin, P. C. et al., Small, 2:485-489 (2006)).
Due to the low intrinsic signal of biomolecules, most existing detection techniques are based on a tagging strategy that links the target molecule to reporters. The number of target molecules is then converted to a specific measure, be it fluorescence signal, radioactivity, or electric current. The ratio between the bound reporters and the target molecules determines the fold of amplification and hence the detection sensitivity of the assay. According to amplification schemes, existing detection techniques can be divided into two categories: direct amplification and enzymatic amplification. In direct amplification, the reporters are conjugated or adsorbed to the surface of detection probes (FIG. 1A). Owing to steric hindrance, the fold of amplification rarely exceeds a few hundreds even with nano- or macro-particle based probes (Nam, 3. M. et al., Science, 301:1884-1886 (2003)). Consequently, the techniques employing direct amplification demand ultrasensitive reporters such as radioisotopes, which often rely on specialized instrument for signal acquisition (Nam, 3. M. et al., Science, 301:1884-1886 (2003); Nam, J. M. et al., J. Am. Chem. Soc., 126:5932-5933 (2004); Chin, C. D. et al., Nat. Med., 17:1015-1019 (2011); Perez, J. M. et al., Nat. Biotechnol. 20:816-820 (2002); Lin, P. C. et al., Small, 2:485-489 (2006); Burtea, C. et al., J. Inorg. Biochem., 99:1135-1144 (2005); Yalow, R. S. and Berson, S. A., The Journal of clinical investigation, 39:1157-1175 (1960); Quinn, Z. A. et al., J. Anal. Atom Spectrom. 17:892-896 (2002); Merkoci, A. et al., Analytical Chemistry, 77:6500-6503 (2005)). In contrast, in enzymatic amplification, the detection probe is linked to an enzyme that can catalyze hydrolysis of chromogenic substrate (FIG. 1B). Enzyme-based amplification has been extensively used in immunosorbent assays and blotting assays for sensitive analyte detection. In particular, enzyme linked immunosorbent assay (ELISA) has been the industrial standard for quantification of protein and other macromolecule since its emergence in 1970s (Engvall, E. and Perlmann, P., Immunochemistry, 8:871-874 (1971); Van Weemen, B. K. and Schuurs, A. H. W., FEBS letters, 15:232-236 (1971)). However, enzyme-substrate interaction is subjected to a number of variables such as enzymatic activity and incubation conditions. Biomolecule quantification with enzyme-linked probes is hindered by the requirement of stringent control, costly calibration as well as the nonlinear nature of enzymatic catalysis.
Therefore, it is an object of the invention to provide methods and compositions for improved methods for detecting target substances.